BIMM110      LECTURES 6-7

THE COMPLEXITY OF THE HUMAN GENOME

Textbook: Pasternak does not have a separate chapter/section for the topics to be covered here;

SLIDES (Lecture 6)

additional information from various sources, e.g. THE HUMAN GENOME PROJECT

 A. The human genome

 - nuclear and mitochondrial genomes
      ~3, 000,000,000 basepairs per haploid nuclear genome; ~2 picograms of DNA per gamete
      16.5 kb mt DNA, hundreds - thousands of copies per cell

- the complexity of a genome; from C_ot analysis: DNA melting and re-association kinetics
- interpretation:
        highly repeated, moderately repeated and unique DNA sequences
        interspersed and tandem repeats;
            SINES (e.g. human Alu sequences) and LINES (e.g. L1 element/Kpn repeat)
            alpha-satellite DNA and telomere repeats
        gene families and superfamilies;       pseudogenes
        exons and introns;
        elements controling gene expression: promoters, enhancers

- minisatellites and microsatellite repeats (a small number of nucleotides (di-, tri, etc.) may be repeated many times; they are flanked by unique DNA sequences)
- variable number of tandem repeat (VNTR) markers

B. Restriction analysis (Review)
concepts to be applied in this course (assumed to be known from preceding lectures in CBB-course):

- restriction enzyme digests, Southern blots, DNA hybridizations with unique and repeated DNA probes,
         
- "zoo blots" ; homologous sequences in other species; cross-hybridization; stringency of hybridization conditions

- restriction fragment length polymorphisms  (RFLPs)
     - use as genetic markers
     - applications to forensics

C. The Polymerase Chain Reaction (PCR)- Review

one of the most important technical inovations in molecular genetics in the past two decades; an understanding of the principles and capability of this methodology is essential for understanding material in future lectures

 1. components of the reaction:
        1) buffer, all four deoxynucleoside triphosphates, thermostable DNA polymerase
        2) template DNA from source of interest
        3) forward and reverse primers (oligonucleotides) defining the DNA segment to be
            amplified
 2. data and detection:
        DNA band on agarose gel after electrophoresis, -  visualized by ethidium bromide;
        should be a single band of expected size
 3. applications
        widely used in forensics (DNA fingerprints)
        very useful for detecting VNTR polymorphisms and trinucleotide expansions in pedigrees
        used in analysis of SNP (single nucleotide polymorphisms)
        used to amplify segments of a gene to check for mutations

RT-PCR:  the first step in the reaction involves reverse transcriptase making cDNA from an mRNA template

 

D. Databases for storing human genome information (see links given on slides)

A. Historical

 - Bernardi, G., Olofsson, B., Filipski, J., Zerial, M., Salinas, J., Cuny, G., Meunier-Rotival, M., and Rodier, F. (1985). The mosaic genome of warm-blooded vertebrates. Science 228, 953-958.
- Britten, R.J., Baron, W.F., Stout, D.B., and Davidson, E.H. (1988). Sources and evolution of human Alu repeated sequences. Proc. Natl. Acad. Sci. USA 85, 4770-4774.
- Hardman, N. (1986). Structure and function of repetitive DNA in eukaryotes. Biochem. J. 234, 1-11.
- Kao, F.T. (1985). Human genome structure. Int. Rev. Cytol. 96, 51-88.
- Manuelidis, L. (1978). Complex and simple sequences in human repeated DNAs. Chromosoma 66, 1-21.
- Nakamura, Y., Leooert, M., O'Connell, P., Wolff, R., Holm, T., Culver, M., Martin, C., Fujimoto, E., Hoff, M., Kumlin, E., and White, R. (1987).      Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235, 1616-1621.
- Singer, M.F. (1982). Highly repeated sequences in mammalian chromosomes. Int. Rev. Cytol. 76, 67-112.
- Willard, H.F. (1985). Chromosome-specific organization of human alpha satellite DNA. Am. J. Hum. Genet. 37, 524-532.
- Willard, H.F., and Waye, J.S. (1987). Hierarchical order in chromosome-specific human alpha satellite DNA. Trends in Genet. 3, 192-198.

 B. (Selected) Recent

 - Hurst, L.D. (1994). The uncertain origin of introns. Nature 371, 381-382.
- Britten, R. J. 1994. Evidence that most human Alu sequences were inserted in a process that ceased about 30 million years ago. Proc. Natl. Acad. Sci. USA 91:6148-6150.
- Brookfield, J. F. Y. 1993. The generation of sequence similarity in SINEs and LINEs. Trends Genet. 9:38
- McMillan, J. P. and M. F. Singer. 1993. Translation of the human LINE-1 element, L1Hs. Proc. Natl. Acad. Sci. USA 90:11533-11537.
- Schmid, C. W. 1993. How many source Alus. Trends in Genet. 9:39
- Jeffreys,A.J. 1987. Highly variable minisatellites and DNA fingerprints. Biochem. Soc. Transac. 15: 309 - 317
- Tyler-Smith, C., and H.F. Willard. 1993. Mammalian chromosome structure. Curr. Opinion Genet. Dev. 3: 390 - 397